DNA: Acelarin is a phosphoramidate prodrug of gemcitabine. Its mechanism of action involves intracellular delivery of gemcitabine monophosphate, which is further phosphorylated to the active triphosphate metabolite (dFdCTP). dFdCTP replaces deoxycytidine during DNA replication, leading to cell cycle arrest and apoptosis. The diphosphate metabolite (dFdCDP) inhibits ribonucleotide reductase (RNR), potentiating the anticancer effect. [1]

Gemcitabine, a nucleoside analog, is frequently used to treat cancer; however, because cancer cells are highly sensitive to drug resistance, its effectiveness is restricted. Gemcitabine is shielded from several important anticancer pathways by the addition of a phosphoramidate motif. A number of prodrugs of gemcitabine phosphoramidate were prepared and tested for their ability to inhibit tumor growth in a variety of tumor cell lines. Of the compounds produced, NUC-1031 had strong effects in vitro.

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Cytostatic Activity: Acelarin (6f) demonstrated potent in vitro cytostatic activity against a range of cancer cell lines. IC50 values (μM) were: 0.035 ± 0.02 in L1210 (murine leukemia), 0.1 ± 0.03 in CEM (human T-lymphocyte), 0.44 ± 0.06 in MiaPaCa-2 (human pancreatic), and 0.15 ± 0.04 in BxPC-3 (human pancreatic). Compared to gemcitabine, Acelarin showed 2- to 4-fold increased potency in the pancreatic cell lines. [1]
Overcoming dCK Resistance: In RT112 cancer cells, the presence of deoxycytidine (a dCK substrate) increased gemcitabine's EC50 75-fold (from 1.4 nM to 104.9 nM). In contrast, Acelarin's activity was not significantly affected (EC50 = 0.2 nM without deoxycytidine vs. 0.7 nM with deoxycytidine), indicating it bypasses dCK-dependent activation. [1]
Overcoming hENT1 Transporter Resistance: In PANC1 pancreatic cancer cells, the nucleoside transport inhibitor dipyridamole markedly reduced gemcitabine's activity (EC50 increased from 611 μM to >2000 μM). Acelarin maintained strong cytotoxicity in the presence of dipyridamole (EC50 = 162 μM vs. 190 μM without inhibitor), demonstrating transporter-independent uptake. [1]
Resistance to Cytidine Deaminase (CDA): UV absorbance spectroscopy showed that gemcitabine was rapidly deaminated within 2 minutes (absorbance shift from 267 nm to 257 nm). Acelarin's absorbance spectrum remained unchanged at 267 nm over 30 minutes, confirming resistance to CDA-mediated degradation. [1]
Intracellular dFdCTP Levels: In BxPC-3 and MiaPaCa-2 cells, Acelarin maintained steady intracellular levels of the active triphosphate metabolite (dFdCTP) even in the presence of dCK inhibitor (2T2D) or hENT1 transporter inhibitor (NBTI). Gemcitabine's dFdCTP levels were significantly reduced under these conditions. [1]
Metabolic Stability in Human Hepatocytes: Acelarin (6f) showed a half-life of 139 minutes in human hepatocyte extracts, indicating mid-range stability suitable for clinical development. [1]
Metabolic Stability in Liver Microsomes: After 1 hour incubation with liver microsomes, 18% of Acelarin remained detectable. [1]

In a pancreatic xenograft model, ProTide significantly reduced tumor size and had less negative effects on body weight than the gemcitabine-treated group, suggesting a superior safety profile. The information clearly indicates that ProTides are stable in the presence of deaminases and do not depend on kinases or nucleoside transporters to function within tumor cells. ProTide NUC-1031 is presently progressing into a Phase I/II clinical investigation and has produced encouraging early efficacy signals, excellent security, and robust pharmacokinetic data showing notable increases in intracellular levels of gemcitabine triphosphate. Phosphoramidate compounds have the potential to be a significant source of novel, highly effective anticancer medications, resulting in a plethora of cutting-edge therapies intended to circumvent cancer resistance mechanisms and enhance patient outcomes [1].

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MiaPaCa-2 Xenograft Model (Pancreatic Cancer): In nude mice bearing MiaPaCa-2 human pancreatic tumors, Acelarin (0.076 mmol/kg, i.p.) significantly reduced tumor volume compared to vehicle control on Day 7 after first administration. The reduction was significantly greater than that achieved with gemcitabine at the same time point. Body weight change was <4% and similar to gemcitabine-treated mice, indicating tolerability. [1]
BxPC-3 Xenograft Model (Gemcitabine-Resistant Pancreatic Cancer): In nude mice bearing BxPC-3 human pancreatic tumors (a gemcitabine-resistant line), Acelarin achieved significant reduction in tumor growth compared to control, while gemcitabine did not. Mean body weight change was less in Acelarin-treated mice than in gemcitabine-treated mice, suggesting better tolerability. [1]
Maximum Tolerated Dose Study: In Balb/c nude mice, Acelarin was administered intraperitoneally at 0.228 mmol/kg (132.3-141.9 mg/kg) twice weekly for 2 weeks, dissolved in 40% Captisol solution. Mice were monitored for body weight change and clinical symptoms. [1]

Carboxypeptidase Y Assay (Activation Pathway): Acelarin (6f) was dissolved in acetone-d6 with Trizma buffer (pH 7.6) and treated with carboxypeptidase Y. ³¹P NMR spectra were recorded over time. Within 10 minutes, a peak at δ 4.35 ppm appeared (intermediate A, lacking ester). A peak at δ 6.80 ppm then appeared and increased over 12 hours, consistent with the stable achiral intermediate (C). This confirmed the proposed activation pathway: ester hydrolysis → spontaneous cyclization (aryl displacement) → hydrolysis → amino acid cleavage to release gemcitabine monophosphate. [1]

Cytotoxicity Assay (MTS): Cancer cell lines (MiaPaCa-2, BxPC-3, L1210, CEM) were seeded in 96-well plates (0.5-100 × 10³ cells/well) and incubated overnight. Cells were then incubated with various concentrations of test compounds for 72 hours. MTS reagent (50 μL) was added, cells were incubated for 4 hours at 37°C, and absorbance was read using a microplate reader to determine IC50 values. [1]
dCK Bypass Assay: RT112 cells were treated with gemcitabine or Acelarin in the presence or absence of deoxycytidine (a dCK substrate). EC50 values were determined by MTT assay measuring viable cells. [1]
Transporter Bypass Assay: PANC1 cells were treated with gemcitabine or Acelarin in the presence or absence of dipyridamole (a nucleoside transport inhibitor). EC50 values were determined as above. [1]
Intracellular dFdCTP Measurement: BxPC-3 and MiaPaCa-2 cells were treated with gemcitabine or Acelarin for 24 hours in the presence or absence of dCK inhibitor (2T2D) or hENT1 inhibitor (NBTI). Cells were washed, lysed, and intracellular dFdCTP levels were quantified by UPLC-MS/MS. [1]
Cytidine Deaminase (CDA) Assay: Gemcitabine and Acelarin were dissolved in assay buffer (50 mM Tris-HCl, pH 7.5) to 100 μM. UV spectra (220-350 nm) were recorded at 25°C. CDA enzyme solution (200 μL) was added, and spectra were recorded at 1-minute intervals for 30 minutes. Degradation was monitored by absorbance shift. [1]

Xenograft Studies:** Female Balb/c nude mice (6-8 weeks old, 20±2 g) were inoculated subcutaneously with MiaPaCa-2 or BxPC-3 human pancreatic cancer cells. When tumors reached appropriate size, mice were randomized into groups. Acelarin and gemcitabine were dissolved in 40% Captisol solution (prepared by dissolving Captisol in pure water, filtered through 0.22 μm filter). Compounds were administered intraperitoneally for 3 weeks. Tumor volumes and body weights were measured regularly. Data were analyzed using Student's t-test. [1]
* **Maximum Tolerated Dose Study:** Female Balb/c nude mice (6-8 weeks old) received intraperitoneal injections of Acelarin (0.228 mmol/kg, 132.3-141.9 mg/kg) or vehicle (40% Captisol) twice weekly for 2 weeks. Mice were monitored daily for body weight change and clinical symptoms. [1]

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Xenograft Studies: Female Balb/c nude mice (6-8 weeks old, 20±2 g) were inoculated subcutaneously with MiaPaCa-2 or BxPC-3 human pancreatic cancer cells. When tumors reached appropriate size, mice were randomized into groups. Acelarin and gemcitabine were dissolved in 40% Captisol solution (prepared by dissolving Captisol in pure water, filtered through 0.22 μm filter). Compounds were administered intraperitoneally for 3 weeks. Tumor volumes and body weights were measured regularly. Data were analyzed using Student's t-test. [1]
Maximum Tolerated Dose Study: Female Balb/c nude mice (6-8 weeks old) received intraperitoneal injections of Acelarin (0.228 mmol/kg, 132.3-141.9 mg/kg) or vehicle (40% Captisol) twice weekly for 2 weeks. Mice were monitored daily for body weight change and clinical symptoms. [1]

Human Serum Stability: Acelarin was incubated with human serum at 37°C and monitored by ³¹P NMR over 13 hours. After 13 hours, 59% of the original compound remained, with 41% converted to metabolite C. Estimated half-life was >14 hours, indicating suitability for clinical studies. [1]
Species-Specific Stability: In rat serum, Acelarin was completely converted to metabolite C within 6.5 minutes (half-life <5 minutes). In dog serum, >80% remained after 14 hours (half-life >100 hours), confirming rapid degradation in rodents due to circulating esterases but stability in other species including humans. [1]
Human Hepatocyte Stability: Acelarin showed a half-life of 139 minutes in human hepatocyte extracts. [1]
Liver Microsome Stability: After 1 hour incubation with liver microsomes, 18% of Acelarin remained detectable. [1]
Intracellular Metabolism: Acelarin delivers gemcitabine monophosphate intracellularly, bypassing the rate-limiting dCK phosphorylation step. It is further metabolized to the active diphosphate (dFdCDP) and triphosphate (dFdCTP) forms. [1]

In Vivo Tolerability (MiaPaCa-2 Model): Body weight change in mice treated with Acelarin was <4% and similar to gemcitabine-treated mice, indicating tolerability. [1]
In Vivo Tolerability (BxPC-3 Model): Mean body weight change was less in Acelarin-treated mice than in gemcitabine-treated mice, suggesting better tolerability. [1]
Maximum Tolerated Dose: Acelarin was well tolerated in mice at 0.228 mmol/kg (132.3-141.9 mg/kg) administered twice weekly for 2 weeks, with no reported deaths or severe clinical symptoms. [1]
Reduced Toxic Metabolite Formation: Acelarin's resistance to CDA degradation suggests it should release less of the harmful deaminated catabolite dFdU, potentially conferring a more favorable safety profile in patients. [1]

[1]. Application of ProTide technology to gemcitabine: a successful approach to overcome the key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development. J Med Chem. 2014 Feb 27;57(4):1531-42.

Background: Acelarin (NUC-1031, compound 6f) is a phosphoramidate prodrug of gemcitabine developed using ProTide technology. It was first patented in 2005 as CPF-3126. The compound is designed to overcome the key resistance mechanisms that limit gemcitabine's efficacy: (1) down-regulation of deoxycytidine kinase (dCK), (2) reduced expression of nucleoside transporters (especially hENT1), and (3) up-regulation of cytidine deaminase (CDA). [1]
Mechanism of Action: Acelarin is taken up into cells independently of nucleoside transporters. The phosphoramidate moiety is metabolized via a proposed pathway: ester hydrolysis (by carboxypeptidase-type enzyme) → spontaneous cyclization (displacing aryl group) → hydrolysis → phosphoramidase cleavage, releasing gemcitabine monophosphate. This bypasses the rate-limiting dCK phosphorylation step. The monophosphate is further phosphorylated to the active diphosphate (dFdCDP, which inhibits RNR) and triphosphate (dFdCTP, which incorporates into DNA causing chain termination). [1]
Clinical Development: Acelarin (NUC-1031) was in clinical development at the time of publication, with a Phase I/II study showing encouraging efficacy signals and a favorable safety profile in patients with advanced solid tumors. The compound demonstrated activity against a range of solid tumors. [1]
Structural Features: Acelarin is an L-alanine benzyl ester phenyl phosphoramidate prodrug of gemcitabine. The phosphoramidate moiety is attached at the 5'-position of the ribose sugar. [1]
Advantage Over Other Prodrugs: Unlike other gemcitabine prodrugs that address only one or two resistance mechanisms, Acelarin overcomes all three key resistance pathways: dCK dependence, transporter dependence, and CDA degradation. [1]

C25H27F2N4O8P

580.4816

580.153

840506-29-8

11169170

White to off-white solid powder

3.616

3

11

12

40

1020

4

C[C@@H](C(=O)OCC1=CC=CC=C1)NP(=O)(OC[C@@H]2[C@H](C([C@@H](O2)N3C=CC(=NC3=O)N)(F)F)O)OC4=CC=CC=C4

NHTKGYOMICWFQZ-KKQYNPQSSA-N

InChI=1S/C25H27F2N4O8P/c1-16(22(33)36-14-17-8-4-2-5-9-17)30-40(35,39-18-10-6-3-7-11-18)37-15-19-21(32)25(26,27)23(38-19)31-13-12-20(28)29-24(31)34/h2-13,16,19,21,23,32H,14-15H2,1H3,(H,30,35)(H2,28,29,34)/t16-,19+,21+,23+,40?/m0/s1

benzyl (2S)-2-[[[(2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4,4-difluoro-3-hydroxyoxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate

GTPL 7389 CPF-31 CPF31Acelarin NUC-1031 NUC 1031 NUC1031GTPL7389 GTPL-7389

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 36 mg/mL (~62.02 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)